1. Field of the Invention
This invention relates to an alkaline proteolytic enzyme, its production by cultivating genetically modified Bacillus strains, and the genetic constructs within the Bacillus strains which code for the alkaline proteolytic enzyme.
2. Description of the Related Art
Proteolytic enzymes are widely used in household laundry detergent formulations to remove protein-based stains from articles of clothing and the like. These enzymes are members of the class known as serine proteases, enzymes which catalyze the cleavage of peptide bonds. To be useful in aqueous detergent solutions, these enzymes must be active at pH values ranging from 7 to 10. They must also be stable to oxidation and enzymatic and non-enzymatic hydrolysis in order to be useful in detergent applications. Various Bacillus strains produce serine proteases under suitable aerobic fermentation conditions. However, the amount of protease made by the wild-type strains and the rate of production of the protease is usually insufficient for commercial production. One method of increasing the protease yield is to generate mutant strains through exposure to radiation or chemical mutagens. However, these classical mutation processes suffer from the disadvantage that they produce only a statistical distribution of the desired mutation and/or that a significant population reverts back to the wild type. In recent years, efforts to overcome these problems have centered on directed genetic modification of bacterial strains. This is accomplished through the introduction of recombinant DNA into host cells under circumstances guaranteeing that the host cells will replicate the recombinant DNA with an increased copy number. In addition, the genes carried by the foreign DNA must be able to be expressed. The genetically engineered organisms do not suffer from the disadvantages often exhibited by classically mutagenized bacteria and are, therefore, often useful for commercial enzyme production.
Methods of making proteases using recombinant DNA technology are known. For example, European Patent EP130756A discloses a method for preparing carbonyl hydrolase (a protease), U.S. Pat. No. 4,760,025 discloses a subtilisin from having an altered amino acid sequence than that produced by the naturally occuring strain. German patent No. DE 2925427 discloses a Bacillus alkaline protease which is highly stable to chelating agents and surfactants, and is produced by fermentation of a Bacillus licheniformis strain. Production of an alkaline proteolytic enzyme by cultivation of B. licheniformis has been described in British patent No. 1,263,765; German patent No. 1,940,488; Russian patent No. 400,116; U.S. Pat. Nos. 3,623,956; 3,623,957.